PARENT SESSION
Posters P2A Type I reaction centres. Abstracts (181-218)

Evidence for bi-directional electron transfer in Chlamydomonas PSI: analysis of mutants of the axial ligands to Chl A₀. Velupillai Mani Ramesh^*,1, Krzysztof Gibasiewicz, Su Lin, Scott Bingham¹, Andrew Webber¹, ¹ School of Life Sciences,Center for the Study of Early Events in Photosynthesis, Tempe, AZ, USA

ABSTRACT- The recent crystal structure of photosystem I (PSI) shows two branches of nearly symmetric cofactors including P700, A, A₀and A₁, bound to the PsaA and PsaB heterodimer. The primary electron acceptor A₀ is a Chl a monomer that could be one or both of two chlorophyll molecules ecA3/ecB3, identified in 2.5 structure. The ecA3/ecB3 Chls are both coordinated by the sulfur atom of methionine 688 of PsaA and 668 of PsaB, respectively. The very symmetrical nature of the ETC chlorophylls raises the question as to wether both ecA3 and ecB3 are involved in electron transfer, or if only a single Chl represents A₀. To address this question we have used site directed mutagenesis to change methionine (axial ligand to Chl A₀) to His, Leu and Ser on both the PsaA and PsaB branches. Replacement of either M (A684) or M (B664) results in significant alterations in growth phenotype. The His and Leu mutants are very light sensitive in the presence of oxygen. Growth is impaired to a greater extent in the B-side mutants. However, all of the mutants are able to grow anaerobically at comparable rates. Ultrafast transient absorbance measurements show that the (A₀^- - A₀) difference signal accumulates in all of the six mutants under neutral condition, indicating that electron transfer between A₀^- and A₁is blocked. Femtosecond transient measurements on a longer time scale up to 4 ns enabled us to resolve a component that decays with a lifetime between 1100 and 1700 ps, and which has a spectrum identical to A₀^-. This indicates that electron transfer from A₀^- to A₁ is slowed from approximately 20 ps (wild type) to 1100 ps in Ser mutants and 1700 ps in His and Leu mutants. The accumulated (A₀^- - A₀) difference signal decay is slowest in the His and Leu mutants where photoautotrophic growth is severely impaired. The observed decay kinetics indicate that the replacement of Met to basic (His), non polar (Leu) and polar (Ser) amino acid residues alters the redox potential of Chl A₀ which in turn alters the electron transfer kinetics between A₀^- and A₁. The results show that both the A- branch and B- branch of the ETC are involved in forward electron transfer at least to A₀ and supports the bidirectional electron transfer in Chlamydomonas PSI.

KEY WORDS: Chlamydomonas, A₀, Photosystem I, Sitedirected mutagenesis